Cancer-related extracellular matrix signatures and related methods and products

ABSTRACT

Proteomic methods for identifying cancer related proteins and related products and kits are provided. The cancer specific proteins are extracellular matrix proteins that are associated with various aspects of cancer. Panels or signature sets of proteins useful in the detection, diagnosis and treatment of cancers as well as monitoring therapeutic progress in a cancer patient are provided herein along with methods for their detection and for their use in targeting imaging and/or therapeutic agents to the tumors via binding to the specified proteins. The proteins were identified using proteomics analyzes of tissue samples taken from cancer patients. In certain aspects the proteins are particularly useful in colon cancer patients.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/389,080, filed on Sep. 29, 2014, which is a national staged filingunder U.S.C. § 371 of PCT International application PCT/US2013/031492,entitled “CANCER-RELATED EXTRACELLULAR MATRIX SIGNATURES AND RELATEDMETHODS AND PRODUCTS,” filed on Mar. 14, 2013, which claims priorityunder 35 U.S.C. § 119(e) to U.S. Provisional Application Ser. No.61/616,981, entitled “EXTRA CELLULAR MATRIX PROTEINS AND CANCER” filedon Mar. 28, 2012, AND to U.S. Provisional Application Ser. No.61/616,987 entitled “CANCER SIGNATURES AND RELATED METHODS AND PRODUCTS”filed on Mar. 28, 2012 which are herein incorporated by reference intheir entirety.

FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under NIH U54-CA126515and U54-CA163109. The Government has certain rights in this invention.

FIELD OF THE INVENTION

The invention relates in some aspects to application of recentlydeveloped proteomic methods for identifying cancer-related extracellularmatrix (ECM) proteins correlated with cancer progression and useful fordiagnosis and prognosis as well as related reagents, products and kits.The cancer-specific extracellular matrix proteins are associated withvarious properties of cancerous tissues. The invention also relates toECM proteins and panels or signature sets of such proteins useful in thedetection, diagnosis and treatment of cancers as well as monitoringtherapeutic progress in a cancer patient. Defined lists of ECM proteinsspecifically expressed in primary tumors and/or in metastases and, insome cases, in metastatic primary tumors but not in non-metastaticprimary tumors are presented. Thus these proteins, both individually andas sets or “signatures” can be used to determine the degree ofmalignancy of tumor biopsies and other samples and can be used to targetspecific binding reagents to tumors for purposes of imaging and therapy.

BACKGROUND OF THE INVENTION

Despite extensive study, cancer, and particularly the process ofmetastasis, remain major causes of illness and mortality. Priorindications suggest that changes in extracellular matrix (ECM) proteinsmay play significant roles in cancer progression and metastasis. Thefield of proteomics involves the study of proteins in complexphysiological systems and their role in these systems. Large data setshave been generated using genomic and proteomic methods, but the use ofthat information to identify the role of extracellular matrix (ECM)proteins in disease has been limited. This is because the ECM isinsoluble and crosslinked and its composition has been difficult todetermine.

SUMMARY OF INVENTION

The invention in some aspects is a method involving administering to asubject having a tumor a binding reagent which interacts specificallywith an extracellular matrix (ECM) protein, wherein the binding reagentis conjugated to an active agent in an effective amount to deliver theactive agent to the tumor. The active agent may be or include adetectable label or a chemotherapeutic agent. The tumor may be, in someembodiments, metastatic.

In some embodiments the ECM protein is a protein selected from an ECMprotein signature characteristic of non-metastatic mammary carcinoma, anECM protein signature characteristic of metastatic mammary carcinoma, anECM protein signature characteristic of mammary carcinoma, an ECMprotein signature characteristic of metastatic primary colon carcinoma,an ECM protein signature characteristic of colon carcinoma metastases,an ECM protein signature characteristic of metastatic colon carcinoma(primary & secondary), and an ECM protein signature whose expression isassociated with colon cancer or metastatic colon cancer.

In other embodiments the ECM protein is an ECM protein characteristic ofmetastatic tumors and is selected from an ECM protein signaturecharacteristic of metastatic mammary carcinoma, an ECM protein signaturecharacteristic of metastatic primary colon carcinoma, an ECM proteinsignature characteristic of colon carcinoma metastases, an ECM proteinsignature characteristic of metastatic colon carcinoma (primary &secondary), and an ECM protein signature whose expression is associatedwith colon cancer or metastatic colon cancer

The binding reagent in other embodiments is an antibody or an antibodyfragment.

The tumor in some embodiments is a tumor of the breast. The ECM proteinmay be selected from an ECM protein signature characteristic ofmetastatic mammary carcinoma. In some embodiments the ECM proteinsignature characteristic of metastatic mammary carcinoma is selectedfrom subgroup 2A, subgroup 2B, and subgroup 2C.

In other embodiments the ECM protein is selected from an ECM proteinsignature characteristic of non-metastatic mammary carcinoma. In someembodiments the ECM protein signature characteristic of non-metastaticmammary carcinoma is a protein selected from subgroup 1A, subgroup 1B,and subgroup 1C.

In some embodiments the ECM protein is a selected from an ECM proteinsignature characteristic of mammary carcinoma. In other embodiments theECM protein signature characteristic of mammary carcinoma is a proteinselected from subgroup 3A, subgroup 3B, and subgroup 3C.

The tumor may be a tumor of the colon. In some embodiments the ECMprotein is selected from an ECM protein signature characteristic ofmetastatic primary colon carcinoma. In other embodiments the ECM proteinsignature characteristic of metastatic primary colon carcinoma is aprotein selected from subgroup 4A, subgroup 4B, and subgroup 4C. In yetother embodiments the ECM protein is selected from an ECM proteinsignature characteristic of colon carcinoma metastases and optionallythe ECM protein signature characteristic of colon carcinoma metastasesis a protein selected from subgroup 5A, subgroup 5B, and subgroup 5C.The ECM protein in other embodiments is selected from an ECM proteinsignature characteristic of metastatic colon carcinoma (primary &secondary). In some embodiments the ECM protein signature characteristicof metastatic colon carcinoma (primary & secondary) is a proteinselected from subgroup 6A, subgroup 6B, and subgroup 6C. In yet otherembodiments the ECM protein is selected from an ECM protein signaturewhose expression is associated with colon cancer or metastatic coloncancer. In some embodiments the ECM protein signature whose expressionis associated with colon cancer or metastatic colon cancer is a proteinselected from subgroup 7A, subgroup 7B, and subgroup 7C.

A composition of a binding reagent which interacts specifically with anextracellular matrix (ECM) protein, wherein the binding reagent isconjugated to an active agent is provided according to other aspects ofthe invention. The ECM protein may be a protein characteristic ofcancers. In some embodiments the binding reagent is an antibody, andoptionally is selected from the group consisting of a monoclonalantibody, a polyclonal antibody, a chimeric antibody, a humanizedantibody, a human antibody, and an antibody fragment. In someembodiments the active agent is a detectable label or a chemotherapeuticagent.

In other embodiments the ECM protein is selected from an ECM proteinsignature characteristic of metastatic mammary carcinoma. In someembodiments the ECM protein signature characteristic of metastaticmammary carcinoma is selected from subgroup 2A, subgroup 2B, andsubgroup 2C. In other embodiments the ECM protein is selected from anECM protein signature characteristic of non-metastatic mammarycarcinoma. In some embodiments the ECM protein signature characteristicof non-metastatic mammary carcinoma is a protein selected from subgroup1A, subgroup 1B, and subgroup 1C. The ECM protein is selected from anECM protein signature characteristic of mammary carcinoma in someembodiments. In other embodiments the ECM protein signaturecharacteristic of mammary carcinoma is a protein selected from subgroup3A, subgroup 3B, and subgroup 3C.

The ECM protein is selected from an ECM protein signature characteristicof metastatic primary colon carcinoma. In some embodiments the ECMprotein signature characteristic of metastatic primary colon carcinomais a protein selected from subgroup 4A, subgroup 4B, and subgroup 4C. Invarious embodiments the ECM protein is selected from an ECM proteinsignature characteristic of colon carcinoma metastases. In someembodiments the ECM protein signature characteristic of colon carcinomametastases is a protein selected from subgroup 5A, subgroup 5B, andsubgroup 5C. The ECM protein, in some embodiments, is selected from anECM protein signature characteristic of metastatic colon carcinoma(primary & secondary). In some embodiments the ECM protein signaturecharacteristic of metastatic colon carcinoma (primary & secondary) is aprotein selected from subgroup 6A, subgroup 6B, and subgroup 6C. Inother embodiments the ECM protein is selected from an ECM proteinsignature whose expression is associated with colon cancer or metastaticcolon cancer. In some embodiments the ECM protein signature whoseexpression is associated with colon cancer or metastatic colon cancer isa protein selected from subgroup 7A, subgroup 7B, and subgroup 7C.

A method is provided, in aspects of the invention, for determining thepresence or absence of one or more ECM proteins characteristic ofmetastatic carcinoma in a tumor and determining whether the canceroustissues are metastatic based on the presence or absence of one or moreECM proteins characteristic of metastatic carcinoma. The method mayfurther comprise treating a subject whose tumor expresses one or more ofthe ECM proteins characteristic of metastatic carcinoma as identifiedherein with an anti-cancer agent, optionally any one of the compositionsdescribed herein. The presence of one more of the ECM proteincharacteristic of metastatic carcinoma in the tumor indicates that thetumor is metastatic, in some embodiments. Two or more ECM proteincharacteristic of metastatic carcinoma may be detected in the tumor.Optionally, five or more ECM protein characteristic of metastaticcarcinoma are detected in the tumor.

In some embodiments the cancer is breast cancer and the ECM protein isselected from an ECM protein signature characteristic of metastaticmammary carcinoma. In some embodiments the ECM protein signaturecharacteristic of metastatic mammary carcinoma is selected from subgroup2A, subgroup 2B, and subgroup 2C.

In other embodiments the tumor is a tumor of the colon. The ECM proteinis selected from an ECM protein signature characteristic of metastaticprimary colon carcinoma in some embodiments. In some embodiments the ECMprotein signature characteristic of metastatic primary colon carcinomais a protein selected from subgroup 4A, subgroup 4B, and subgroup 4C. Inother embodiments the ECM protein is selected from an ECM proteinsignature characteristic of colon carcinoma metastases. In someembodiments the ECM protein signature characteristic of colon carcinomametastases is a protein selected from subgroup 5A, subgroup 5B, andsubgroup 5C. The ECM protein, in other embodiments, is selected from anECM protein signature characteristic of metastatic colon carcinoma(primary & secondary). In some embodiments the ECM protein signaturecharacteristic of metastatic colon carcinoma (primary & secondary) is aprotein selected from subgroup 6A, subgroup 6B, and subgroup 6C. Inother embodiments the ECM protein is selected from an ECM proteinsignature whose expression is associated with colon cancer or metastaticcolon cancer. In some embodiments the ECM protein signature whoseexpression is associated with colon cancer or metastatic colon cancer isa protein selected from subgroup 7A, subgroup 7B, and subgroup 7C.

Optionally, the method may involve determining whether an ECM proteincharacteristic of non-metastatic carcinoma is present in the tumor. Insome embodiments the presence of one or more of the ECM proteinscharacteristic of non-metastatic carcinoma in the tumor indicates thatthe tumor is not metastatic. In other embodiments the ECM protein isselected from an ECM protein signature characteristic of non-metastaticmammary carcinoma. In some embodiments the ECM protein signaturecharacteristic of non-metastatic mammary carcinoma is a protein selectedfrom subgroup 1A, subgroup 1B, and subgroup 1C.

Optionally, the tumor may be an isolated tumor sample from a subject.

In some embodiments a level of expression of the ECM proteincharacteristic of metastatic carcinoma is measured and is at least twicea baseline level.

In other embodiments the ECM protein characteristic of metastaticcarcinoma is detected by immunohistochemistry.

The method may be a method of tracking the progression of a tumor to ametastatic state by measuring the presence or absence of one or more ECMprotein characteristic of metastatic carcinoma in the tumor over time.

Alternatively, the method may involve determining whether or not asubject has metastatic cancer comprising, determining the presence orabsence of a signature of ECM proteins whose expression is associatedwith metastatic cancer in an isolated tissue sample from a subject anddetermining whether the subject has metastatic cancer based on thepresence or absence of the signature of ECM proteins, such that when thesignature of ECM proteins is present in the isolated tissue sample thesubject has metastatic cancer.

A method for monitoring progression of a tumor to a metastatic state isprovided in other aspects of the invention. The method involvesmeasuring the presence or absence of a signature of ECM proteins intissue samples isolated from the subject at a first time point and asecond time point and determining the progression of the tumor to ametastatic state based on changes in the presence or absence of thesignature of ECM proteins at the first and second time points. Themethod may also involve treating a subject having a signature of ECMproteins as identified herein with an anti-cancer agent, optionally anyone of the compositions described herein.

In some embodiments the cancer is colon cancer. The signature of ECMproteins, in some embodiments, includes at least 2, at least 5, at least10, at least 15, at least 20, at least 25, at least 30 or all of theproteins selected from an ECM protein signature whose expression isassociated with colon cancer or metastatic colon cancer. In someembodiments the ECM protein signature whose expression is associatedwith colon cancer or metastatic colon cancer is a protein selected fromsubgroup 7A, subgroup 7B, and subgroup 7C. In other embodiments thesignature of ECM proteins includes at least 2, at least 5, at least 10,at least 15, at least 20, at least 25, at least 30 or all of theproteins selected from an ECM protein signature characteristic ofmetastatic colon carcinoma (primary & secondary). In some embodimentsthe ECM protein signature characteristic of metastatic colon carcinoma(primary & secondary) is a protein selected from subgroup 6A, subgroup6B, and subgroup 6C. In yet other embodiments the signature of ECMproteins includes at least 2, at least 5, at least 10, at least 15, atleast 20, at least 25, at least 30 or all of the proteins selected froman ECM protein signature characteristic of colon carcinoma metastases.In some embodiments the ECM protein signature characteristic of coloncarcinoma metastases is a protein selected from subgroup 5A, subgroup5B, and subgroup 5C. In other embodiments the signature of ECM proteinsincludes at least 2, at least 5, at least 10, at least 15, at least 20,at least 25, at least 30 or all of the proteins selected from an ECMprotein signature characteristic of metastatic primary colon carcinoma.In some embodiments the ECM protein signature characteristic ofmetastatic primary colon carcinoma is a protein selected from subgroup4A, subgroup 4B, and subgroup 4C.

Alternatively, the cancer may be breast cancer. In some embodiments thesignature of ECM proteins includes at least 2, at least 5, at least 10,at least 15, at least 20, at least 25, at least 30 or all of theproteins selected from the group consisting of an ECM protein signaturecharacteristic of metastatic mammary carcinoma. In other embodiments theECM protein signature characteristic of metastatic mammary carcinoma isselected from subgroup 2A, subgroup 2B, and subgroup 2C.

In yet other embodiments the signature of ECM proteins includes at least5, at least 15, or at least 30 of the selected proteins.

The signature of ECM proteins may be a signature characteristic ofmetastatic carcinoma. In some embodiments when one or more of the ECMproteins characteristic of the signature of metastatic carcinoma ispresent at a higher level in the isolated tissue samples from the secondtime point the tumor has progressed to a metastatic state. In otherembodiments when the signature of ECM protein characteristic ofmetastasis is present at a lower level in the isolated tissue samplesfrom the second time point the tumor has regressed to a less metastaticstate.

The method may further comprise the step of determining a level ofexpression of a signature of primary ECM proteins in the isolated tissuesamples and comparing the levels of the signature of primary ECMproteins to the levels of a signature of metastasis ECM proteins.

In some embodiments the method involves administering a chemotherapeuticagent to the subject before the tissue sample is isolated from thesubject at the second time point.

In other embodiments the proteins may be detected using one or moreantibodies that specifically bind to the proteins using a massspectrometry method and/or a chromatographic method or animmunohistochemical technique. The proteins are analyzed using aquantitative ELISA in other embodiments.

A kit is provided in other aspects of the invention. The kit includes aset of binding reagents which interacts specifically with a signature ofECM proteins whose expression is associated with metastatic colon orbreast cancer; a set of reagents for performing an immunohistochemistryreaction using the set of binding reagents; and instructions forperforming the immunohistochemistry reactions.

In some embodiments the set of binding reagents interacts specificallywith at least 2, at least 5, at least 10, at least 15, at least 20, atleast 25, at least 30 or all of the proteins selected from an ECMprotein signature whose expression is associated with colon cancer ormetastatic colon cancer. In some embodiments the ECM protein signaturewhose expression is associated with colon cancer or metastatic coloncancer is a protein selected from subgroup 7A, subgroup 7B, and subgroup7C.

In some embodiments the set of binding reagents interacts specificallywith at least 2, at least 5, at least 10, at least 15, at least 20, atleast 25, at least 30 or all of the proteins selected from an ECMprotein signature characteristic of metastatic colon carcinoma (primary& secondary), and instructions include instructions for identifying thepresence of ECM proteins characteristic of metastatic colon carcinoma(primary and secondary). In some embodiments the ECM protein signaturecharacteristic of metastatic colon carcinoma (primary & secondary) is aprotein selected from subgroup 6A, subgroup 6B, and subgroup 6C.

In other embodiments the set of binding reagents interacts specificallywith at least 2, at least 5, at least 10, at least 15, at least 20, atleast 25, at least 30 or all of the proteins selected from an ECMprotein signature characteristic of colon carcinoma metastases, andwherein the instructions include instructions for identifying thepresence of ECM proteins characteristic of colon carcinoma metastases.In some embodiments the ECM protein signature characteristic of coloncarcinoma metastases is a protein selected from subgroup 5A, subgroup5B, and subgroup 5C.

The set of binding reagents interacts specifically with at least 2, atleast 5, at least 10, at least 15, at least 20, at least 25, at least 30or all of the proteins selected from an ECM protein signaturecharacteristic of metastatic primary colon carcinoma, and wherein theinstructions include instructions for identifying the presence of ECMprotein characteristic of metastatic primary colon carcinoma in otherembodiments. In some embodiments the ECM protein signaturecharacteristic of metastatic primary colon carcinoma is a proteinselected from subgroup 4A, subgroup 4B, and subgroup 4C.

In yet other embodiments the set of binding reagents interactsspecifically with at least 2, at least 5, at least 10, at least 15, atleast 20, at least 25, at least 30 or all of the proteins selected froman ECM protein signature characteristic of metastatic mammary carcinomaand wherein the instructions include instructions for identifying thepresence of ECM protein characteristic of metastatic mammary carcinoma.In some embodiments the ECM protein signature characteristic ofmetastatic mammary carcinoma is selected from subgroup 2A, subgroup 2B,and subgroup 2C.

In some embodiments the set of binding reagents interacts specificallywith at least 2, at least 5, at least 10, at least 15, at least 20, atleast 25, at least 30 or all of the proteins selected from an ECMprotein signature characteristic of mammary carcinoma, and wherein theinstructions include instructions for identifying the presence of ECMprotein characteristic of mammary carcinoma. In some embodiments the ECMprotein signature characteristic of mammary carcinoma is a proteinselected from subgroup 3A, subgroup 3B, and subgroup 3C.

The set of binding reagents interacts specifically with at least five,at least fifteen, or at least thirty of the selected proteins, in someembodiments.

In other aspects the invention is a method, involving administering to asubject having a tumor an extracellular matrix (ECM) protein inhibitor,wherein the ECM protein inhibitor is an inhibitor of a protein selectedfrom any of the proteins of an ECM protein signature characteristic ofnon-metastatic mammary carcinoma, an ECM protein signaturecharacteristic of metastatic mammary carcinoma, an ECM protein signaturecharacteristic of mammary carcinoma, an ECM protein signaturecharacteristic of metastatic primary colon carcinoma, an ECM proteinsignature characteristic of colon carcinoma metastases, an ECM proteinsignature characteristic of metastatic colon carcinoma (primary &secondary), and an ECM protein signature whose expression is associatedwith colon cancer or metastatic colon cancer.

In some embodiments the ECM protein is characteristic of metastaticcarcinoma and is selected from an ECM protein signature characteristicof metastatic mammary carcinoma, an ECM protein signature characteristicof metastatic primary colon carcinoma, an ECM protein signaturecharacteristic of colon carcinoma metastases, an ECM protein signaturecharacteristic of metastatic colon carcinoma (primary & secondary), andan ECM protein signature whose expression is associated with coloncancer or metastatic colon cancer.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

DETAILED DESCRIPTION

Cancer is a complex disease, progressing from initiation of a primarytumor, typically via one or more mutational events leading to excessivegrowth of a primary tumor. So long as the primary tumor remainsrestricted to its site of origin, it is often possible to resect itsurgically and it is often referred to as a benign tumor. However,further changes, both intrinsic to the tumor cells themselves and byrecruitment of non-tumor cells that interact with the tumor cells (oftencalled “stromal” cells), lead to progression in the state of the tumoreventually resulting in invasion into underlying tissues. Such tumorsare referred to as invasive or malignant and are much harder to treat bysurgery because it is difficult to ensure removal of all the tumor cellssince they have invaded and spread. This problem is exacerbated by theprocess of metastasis, in which tumor cells detach from the primarytumor and migrate away from it, eventually penetrating into thevasculature and/or the lymphatics and spreading extensively through thebody seeding secondary tumors or metastases at distant sites. Althoughsome metastases can be removed surgically, they are frequently numerous,may be small and hard to detect and are therefore impossible to removeby surgery. Treatment then calls for radiotherapy or chemotherapy and,despite decades of effort, these methods remain ineffective andmetastatic cancer is responsible for 90% of cancer deaths. Therefore,there is a pressing need for better understanding of the mechanisms ofmetastasis and the development of methods to detect and eradicatemetastases.

The invention described herein offers several approaches to tacklingthese challenges. The focus is on a particular compartment of tumorsknown as the extracellular matrix (ECM), a complex meshwork of proteinfibrils with associated growth factors and enzymes that providesmultiple stimuli for tumor and stromal cells promoting cellproliferation and cell survival and providing substrates for cellmigration and invasion and protective niches for tumor cells. Theseoften provide survival signals to the tumor cells in the face of immuneor therapeutic attack. The biochemical composition of the ECM has beendifficult to define because of its very nature as a meshwork ofinsoluble and crosslinked fibrils that render standard biochemicalanalyses impossible.

The methods used according to the invention employ very recent advancesin technology combining selective enrichment of the ECM, bioinformaticdefinition of the complete inventory of ECM proteins encoded in thegenome and application of mass spectrometry. These methods have madepossible the determination of the ECM complement of any tissue fromsmall samples of tissue. This approach has revealed the striking resultthat any tissue contains ˜150 ECM proteins and that each tissue ischaracterized by a specific set of ECM proteins—an ECM signature.Furthermore, the ECM signatures of primary tumors differ from those ofsurrounding normal tissue and those of non-metastatic primary tumorsdiffer from those of closely related metastatic primary tumors andmetastases differ from their parent primaries. Thus, it is possible todefine ECM signatures characteristic of tumors in different stages ofmalignancy and metastatic progression. Application of these methods tohuman tumor samples, including specifically those of mammary and coloncarcinomas has defined a series of ECM signatures characteristic ofdifferent stages in the progression of these tumors. Thus, the inventionin some aspects relates to the application of new methods combiningbioinformatics and mass spectrometry to characterize and compare the ECMcompositions of normal human tissues, primary tumors and metastases.

We describe several methods based on application and exploitation ofthese signatures and the proteins therein. Methods for determining these“signatures” from patient samples and thus providing evidence as towhether those patient samples are “non-metastatic primary tumor,” or“metastatic primary tumor” or “metastases”. Such methods are based onthe signatures defined by mass spectrometry (examples of which arelisted herein) and are determined using specific sets of antibodies orsimilar specific probe reagents to define which proteins of thesignatures are present in the samples. Methods includeimmunohistochemistry, ELISA, protein arrays and similar methods. Use ofsuch methods provides diagnostic, prognostic and monitoring informationallowing improved management of cancer patients' care and therapy.

Methods for targeting imaging agents (e.g., radionuclides, fluorescentreporters or other detectable reagents, etc.) specifically to individualproteins within the “signatures” to detect the location, extent andprogression of primary tumors and/or metastases are also encompassedwithin the invention. Targeting may be by use of specific antibodies orother specific binding reagents tagged with such imaging agents.

Methods for targeting therapeutic agents (e.g., radionuclides,chemotherapeutic drugs, toxins, cytokines, etc.) specifically toindividual proteins within the “signatures” in order to concentrate suchtherapeutic agents to primary tumors and/or metastases and thus improvetherapeutic index are also aspects of the invention. Targeting of thetherapeutic agents may be achieved by attaching them to specific bindingreagents such as antibodies against ECM proteins defined in the aspectsdescribed above. Other aspects of the invention relate to methods fortargeting functions of specific ECM proteins using specific reagentsdesigned to interfere with their pro-metastatic functions. Exampleswould include blocking the interactions of ECM proteins with theirreceptors or with other ECM proteins (using antibodies, antibodyfragments, peptides or peptidomimetics) and, in the case of ECM orECM-associated proteins with enzymatic activities, inhibitors of thoseactivities.

One method of the invention involves the use of signatures of ECMproteins to identify and/or characterize tissue. The signatures of ECMproteins can be used to identify the presence or absence of a type oftissue, such as cancerous tissue in a sample or in a body. For instance,a tissue sample may be isolated from the body of a subject and thesignature of ECM proteins can be examined.

As used herein, an isolated tissue sample is tissue obtained from atissue biopsy, a surgically resected tumor, or any other tumor massremoved from the body using methods well known to those of ordinaryskill in the related medical arts. The tissue may be known to becancerous or suspected of being cancerous. The phrase “suspected ofbeing cancerous” as used herein means a cancer tissue sample believed byone of ordinary skill in the medical arts to contain cancerous cells.Methods for obtaining the sample from a biopsy include grossapportioning of a mass, microdissection, laser-based microdissection, orother art-known cell-separation methods. The tissue may also be ahistological section.

Because of the variability of the cell types in diseased-tissue biopsymaterial, and the variability in sensitivity of the predictive methodsused, the sample size required for analysis may be 5 mg, 10 mg, 15 mg,25 mg, 30 mg, or 50 mg or greater of tissue. Alternatively it may beportions or sections of biopsy sized tissue samples. The appropriatesample size may be determined based on the cellular composition andcondition of the biopsy and the standard preparative steps for thisdetermination and subsequent isolation of the proteins for use in theinvention are well known to one of ordinary skill in the art.

Alternatively, the signature of ECM proteins may be analyzed directly ina body. For instance, one or more or a panel of binding reagents such assmall molecules, binding peptides or antibodies capable of recognizingone or more or the signature of ECM proteins may be administered to thesubject directly. The binding reagents may be labeled in order to assistwith visualization of the ECM. Alternatively, other reagents may be usedto provide visualization of the ECM. In other embodiments the tissuesmay be removed from the subject before exposure to the binding reagents.

An ECM protein, as used herein, refers to any protein recognized asbeing a part of the ECM. ECM is a fundamental and important component ofmetazoan organisms providing architectural support and anchorage for thecells. The ECM consists of a complex meshwork of highly cross-linkedproteins and exists as interstitial forms within organs and asspecialized forms, such as basement membranes underlying epithelia,vascular endothelium, and surrounding certain other tissues and celltypes (e.g. neurons, muscles). Cells adhere to the ECM via transmembranereceptors, among which integrins are the most prominent. Thesecell-matrix interactions result in the stimulation of various signalingpathways controlling proliferation and survival, differentiation,migration, etc. The composition of the ECM and the repertoire of ECMreceptors determine the responses of the cells. The biophysicalproperties of the ECM (deformability or stiffness) have also been shownto modulate these cellular functions. In addition to core ECM components(fibronectins, collagens, laminins, proteoglycans, etc.), the ECM servesas a reservoir for growth factors and cytokines and ECM-remodelingenzymes that collaborate with ECM proteins to signal to the cells.

The methods involve the use of a ECM protein or a signature of ECMproteins. A signature of ECM proteins, as used herein, refers to a setof information useful for identifying or characterizing a tissue. Theset of information includes 2 or more ECM proteins associated with thattissue. The signature or ECM proteins can be used to identify tissues ina tissue sample or it may be used to characterize the tissue. Forinstance the ECM protein binding reagents or signature of ECM proteinsmay be used to identify the metastatic potential of a tissue. It ispossible to identify the degree of malignancy of a tissue by comparingdifferent signatures of ECM proteins. For instance a method involvingthe screening of set of normal ECM proteins versus a set of ECM proteincharacteristic of metastatic carcinoma would provide information such asa weighted value for the degree of metastasis of the tissue such thatthe skilled artisan can determine whether a tissue is metastatic or not.Such information can be characterized in terms of a sliding scale ofmetastatic potential.

ECM protein binding reagents may be developed for tissues havingdifferent properties (ie. highly or poorly metastatic).

For instance, provided herein as part of the invention is a set of ECMproteins (i.e., a signature) characteristic of non-metastatic mammarycarcinomas and a set of ECM proteins characteristic of metastaticmammary carcinoma. The ECM protein signature characteristic ofnon-metastatic carcinoma comprises proteins which are characteristic ofcancerous tissues that are not metastatic, have not yet metastasized orhave low metastatic potential. The ECM protein signature characteristicof non-metastatic carcinoma can be used to identify or characterizecancerous tissues that are non- or poorly metastatic. The ECM proteinsignature characteristic of non-metastatic mammary carcinoma of theinvention is referred to as GROUP 1 and includes but is not limited toany of the following proteins: COL17A1, COL19A1, Emilin2, Itih3, LAMA2,PLAT (tPA), THBS2, TNXB, Aspn, Col28a1, COL6A6, EMID2, FBLN5, FLG2,Gm7455, Hmcn1, Lum, MMP1, MMP19, NGLY1, OGN, PRELP, S100A16, S100A8,Timp3, Vwa1, COL25A1, HRNR, ITIH5, and MMP2. In some embodiments, theECM protein signature characteristic of non-metastatic mammary carcinomadoes not include MMP2 and, or S100A8.

In other embodiments the ECM protein signature characteristic ofnon-metastatic mammary carcinoma is referred to as subgroup 1A andincludes 1 or more of the following proteins: COL17A1, COL19A1, Emilin2,Itih3, LAMA2, PLAT (tPA), THBS2, TNXB, Aspn, Col28a1, COL6A6, EMID2,FBLN5, FLG2, Gm7455, Hmcn1, Lum, MMP1, MMP19, NGLY1, OGN, PRELP,S100A16, S100A8, Timp3, and Vwa1. In some embodiments, the ECM proteinsignature characteristic of non-metastatic mammary carcinoma does notinclude S100A8.

In other embodiments the ECM protein signature characteristic ofnon-metastatic mammary carcinoma is referred to as subgroup 1B andincludes 1 or more of the following proteins: COL17A1, COL19A1, Emilin2,Itih3, LAMA2, PLAT (tPA), THBS2, and TNXB.

In other embodiments the ECM protein signature characteristic ofnon-metastatic mammary carcinoma is referred to as subgroup 1C andincludes 1 or more of the following proteins: Aspn, Col28a1, COL6A6,EMID2, FBLN5, FLG2, Gm7455, Hmcn1, Lum, MMP1, MMP19, NGLY1, OGN, PRELP,S100A16, S100A8, Timp3, and Vwa1. In some embodiments, the ECM proteinsignature characteristic of non-metastatic mammary carcinoma does notinclude S100A8.

The ECM proteins characteristic of metastatic mammary carcinoma areproteins which are characteristic of cancerous tissues that aremetastatic or have high metastatic potential. These ECM proteinscharacteristic of metastatic carcinoma can be used to identify orcharacterize cancerous tissue that is metastatic or highly metastatic.The ECM protein signature characteristic of metastatic mammary carcinomaof the invention is referred to as GROUP 2 and includes but is notlimited to any of the following proteins: C1qc, CTSB, CTSF, EGLN1, F10,F13b, LTBP3, S100A2, SNED1, SRPX, TIMP1, Vwf, ADAM10, ADAM9, AGRN,ANGPTL4, C1qa, COL22A1, COL24A1, COL4A6, CST3, CTGF, CTSC, EFEMP2,Habp2, HCFC2, HTRA1, IGFBP4, Il16, Itih4, LEPREL2, LOXL2, MFGE8, P4HTM,Papln, Plxnb2, PRG4, S100A10, Serpina1b, SERPINC1, SERPINE2, Serpinf1,TINAGL1, BMP1, COL23A1, CYR61, LAMC2, LOX, SERPINA1, WNT5B. In someembodiments the ECM proteins characteristic of metastatic mammarycarcinoma are not one or more of: CTSB, ANGPTL4, CTGF, LOXL2, CYR61,LOX.

In other embodiments the ECM protein characteristic of metastaticmammary carcinoma is referred to as subgroup 2A and includes 1 or moreof the following proteins: C1qc, CTSB, CTSF, EGLN1, F10, F13b, LTBP3,S100A2, SNED1, SRPX, TIMP1, Vwf, ADAM10, ADAM9, AGRN, ANGPTL4, C1qa,COL22A1, COL24A1, COL4A6, CST3, CTGF, CTSC, EFEMP2, Habp2, HCFC2, HTRA1,IGFBP4, Il16, Itih4, LEPREL2, LOXL2, MFGE8, P4HTM, Papln, Plxnb2, PRG4,S100A10, Serpina1b, SERPINC1, SERPINE2, Serpinf1, and TINAGL1. In someembodiments the ECM proteins characteristic of metastatic mammarycarcinoma are not one or more of: CTSB, ANGPTL4, CTGF, LOXL2.

In other embodiments the ECM proteins characteristic of metastaticmammary carcinoma are referred to as subgroup 2B and include 1 or moreof the following proteins: C1qc, CTSB, CTSF, EGLN1, F10, F13b, LTBP3,S100A2, SNED1, SRPX, TIMP1, and Vwf. In some embodiments the ECM proteincharacteristic of metastatic mammary carcinoma is not CTSB.

In other embodiments the ECM protein signature characteristic ofmetastatic mammary carcinoma is referred to as subgroup 2C and includes1 or more of the following proteins: ADAM10, ADAM9, AGRN, ANGPTL4, C1qa,COL22A1, COL24A1, COL4A6, CST3, CTGF, CTSC, EFEMP2, Habp2, HCFC2, HTRA1,IGFBP4, Il16, Itih4, LEPREL2, LOXL2, MFGE8, P4HTM, Papln, Plxnb2, PRG4,S100A10, Serpina1b, SERPINC1, SERPINE2, Serpinf1, and TINAGL1. In someembodiments the ECM protein characteristic of metastatic mammarycarcinoma are not one or more of: ANGPTL4, CTGF, LOXL2.

In yet other embodiments the ECM protein signature characteristic ofmammary carcinoma comprises one or more proteins included in any of thegroups above. The ECM protein signature characteristic of mammarycarcinoma of the invention is referred to as GROUP 3 and includes but isnot limited to any of the following proteins: COL17A1, COL19A1, Emilin2,Itih3, LAMA2, PLAT (tPA), THBS2, TNXB, Aspn, Col28a1, COL6A6, EMID2,FBLN5, FLG2, Gm7455, Hmcn1, Lum, MMP1, MMP19, NGLY1, OGN, PRELP,S100A16, S100A8, Timp3, Vwa1, COL25A1, HRNR, ITIH5, MMP2, C1qc, CTSB,CTSF, EGLN1, F10, F13b, LTBP3, S100A2, SNED1, SRPX, TIMP1, Vwf, ADAM10,ADAM9, AGRN, ANGPTL4, C1qa, COL22A1, COL24A1, COL4A6, CST3, CTGF, CTSC,EFEMP2, Habp2, HCFC2, HTRA1, IGFBP4, Il16, Itih4, LEPREL2, LOXL2, MFGE8,P4HTM, Papln, Plxnb2, PRG4, S100A10, Serpina1b, SERPINC1, SERPINE2,Serpinf1, TINAGL1, BMP1, COL23A1, CYR61, LAMC2, LOX, SERPINA1, WNT5B. Insome embodiments the mammary ECM protein is not one or more of: S100A8,MMP2, CTSB, ANGPTL4, CTGF, LOXL2, CYR61, LOX.

In other embodiments the ECM protein signature characteristic of mammarycarcinoma is referred to as subgroup 3A and includes 1 or more of thefollowing proteins: COL17A1, COL19A1, Emilin2, Itih3, LAMA2, PLAT (tPA),THBS2, TNXB, Aspn, Col28a1, COL6A6, EMID2, FBLN5, FLG2, Gm7455, Hmcn1,Lum, MMP1, MMP19, NGLY1, OGN, PRELP, S100A16, S100A8, Timp3, Vwa1, C1qc,CTSB, CTSF, EGLN1, F10, F13b, LTBP3, S100A2, SNED1, SRPX, TIMP1, Vwf,ADAM10, ADAM9, AGRN, ANGPTL4, C1qa, COL22A1, COL24A1, COL4A6, CST3,CTGF, CTSC, EFEMP2, Habp2, HCFC2, HTRA1, IGFBP4, Il16, Itih4, LEPREL2,LOXL2, MFGE8, P4HTM, Papln, Plxnb2, PRG4, S100A10, Serpina1b, SERPINC1,SERPINE2, Serpinf1, and TINAGL1. In some embodiments the mammary ECMproteins of subgroup 3A are not one or more of S100A8, CTSB, ANGPTL4,CTGF, LOXL2.

In other embodiments the ECM protein signature characteristic of mammarycarcinoma is referred to as subgroup 3B and includes 1 or more of thefollowing proteins: COL17A1, COL19A1, Emilin2, Itih3, LAMA2, PLAT (tPA),THBS2, TNXB, C1qc, CTSB, CTSF, EGLN1, F10, F13b, LTBP3, S100A2, SNED1,SRPX, TIMP1, and Vwf. In some embodiments the mammary ECM protein ofsubgroup 3B is not S100A8.

In other embodiments the ECM protein signature characteristic of mammarycarcinoma is referred to as subgroup 3C and includes 1 or more of thefollowing proteins: Aspn, Col28a1, COL6A6, EMID2, FBLN5, FLG2, Gm7455,Hmcn1, Lum, MMP1, MMP19, NGLY1, OGN, PRELP, S100A16, S100A8, Timp3,Vwa1, ADAM10, ADAM9, AGRN, ANGPTL4, C1qa, COL22A1, COL24A1, COL4A6,CST3, CTGF, CTSC, EFEMP2, Habp2, HCFC2, HTRA1, IGFBP4, Il16, Itih4,LEPREL2, LOXL2, MFGE8, P4HTM, Papln, Plxnb2, PRG4, S100A10, Serpina1b,SERPINC1, SERPINE2, Serpinf1, and TINAGL1. In some embodiments themammary ECM proteins of subgroup 3C are not one or more of S100A8,ANGPTL4, CTGF, LOXL2.

Also provided herein is a signature of ECM proteins whose expression isassociated with metastatic colon carcinoma. This signature is a set ofECM proteins that specifically identifies cancerous tissue in a subjector that has been isolated from a subject that has colon carcinoma. Thissignature specifically identifies these tissues because the ECM proteinswithin this signature are present in any of the cancerous tissues from apatient with metastases (primary tumor, secondary tumor- or metastases-,or both), and are not present on a normal non-cancerous tissues.

An exemplary signature of ECM proteins whose expression is associatedwith colon cancer or metastatic colon cancer is at least 1 or 2 proteinsidentified below as being an ECM protein signature characteristic ofmetastatic primary colon carcinoma, an ECM protein signaturecharacteristic of colon carcinoma metastases, or an ECM proteinsignature characteristic of metastatic colon carcinoma (primary &secondary), referred as GROUP 7, and comprising 1 or more of thefollowing proteins: ADAM10, CLEC11A, FGL2, LOXL1, MFGE8, MMP9, MMRN1,MUC13, PAPLN, PLOD3, PLXNB2, S100A16, ADAM9, ADAMTSL1, C15orf44, CSPG4,EMID1, FCN1, FMOD, LEFTY1, LEPRE1, MEGF8, MMP1, MMP11, MMP12, MMP2,MMRN2, PLOD2, PLXDC2, PLXND1, S100A11, S100A14, SERPINA3, SERPINF1,SPARC, SVEP1, TGFB1, ADAMDEC1, ANXA13, ANXA7, COL8A1, GREM1, INHBE,LEFTY2, LTBP3, LTBP4, MATN2, MFAP4, MUC5B, THSD4, COMP, HPX, IGFALS,SPP1, BMP1, C1QTNF5, FNDC1, ANXA3, C1QC, CCL21, COL10A1, COL15A1,COL27A1, CRLF3, CTSA, CTSH, CXCL12, F12, FCN3, HABP2, IL16, ITIH2, KNG1,LAMA3, LEFTY1, MMP12, MMP7, MST1, NID1, PCOLCE, PRG4, S100A12, S100A4,S100P, SERPINA1, SERPINA3, SERPINB5, SERPINB6, SERPINB9, SERPINC1,SERPIND1, SERPING1, THBS1, ANXA5, HCFC1, HTRA1, LTBP2, MXRA5, ST14,COL22A1, GPC4, LOXL2, MUC16, PXDN, SFTPD, THBS2, C1QA, C1QB, F2, PLOD1,SERPINB1, TIMP1, AGRN, EFEMP2, HMCN1, SERPINE2, A2M, AEBP1, ANXA1,ANXA11, ANXA4, ANXA6, COL18A1, COL4A1, CSTB, CTSB, CTSD, ECM1, ELANE,F9, FCN1, HRG, IGFBP7, ITIH1, ITIH4, LAMB3, LAMC2, LGALS9, LMAN1, PLG,S100A11, S100A6, S100A8, S100A9, SERPINF1, and SERPINH1. In someembodiments, the ECM protein signature characteristic of colon carcinomadoes not include one or more of: MMP9, MMP2, SPARC, TGFB1, SPP1, LOXL2,CTSB and/or S100A8.

In other embodiments the ECM protein signature characteristic of coloncarcinoma is referred to as subgroup 7A and includes 1 or more of thefollowing proteins: ADAM10, CLEC11A, FGL2, LOXL1, MFGE8, MMP9, MMRN1,MUC13, PAPLN, PLOD3, PLXNB2, S100A16, ADAM9, ADAMTSL1, C15orf44, CSPG4,EMID1, FCN1, FMOD, LEFTY1, LEPRE1, MEGF8, MMP1, MMP11, MMP12, MMP2,MMRN2, PLOD2, PLXDC2, PLXND1, S100A11, S100A14, SERPINA3, SERPINF1,SPARC, SVEP1, TGFB1, COMP, HPX, IGFALS, SPP1, BMP1, C1QTNF5, FNDC1,ANXA5, HCFC1, HTRA1, LTBP2, MXRA5, ST14, COL22A1, GPC4, LOXL2, MUC16,PXDN, SFTPD, and THBS2. In some embodiments the colon carcinoma ECMprotein signature does not include one or more of: MMP9, MMP2, SPARC,TGFB1, SPP1, LOXL2.

In other embodiments, the ECM protein signature characteristic of coloncarcinoma is referred to as subgroup 7B and includes 1 or more of thefollowing proteins: ADAM10, CLEC11A, FGL2, LOXL1, MFGE8, MMP9, MMRN1,MUC13, PAPLN, PLOD3, PLXNB2, S100A16, COMP, HPX, IGFALS, SPP1, ANXA5,HCFC1, HTRA1, LTBP2, MXRA5, and ST14. In some embodiments the coloncarcinoma ECM protein is not MMP9 and/or SP 1.

In other embodiments, the ECM protein signature characteristic of coloncarcinoma is referred to as subgroup 7C and includes 1 or more of thefollowing proteins: ADAM9, ADAMTSL1, C15orf44, CSPG4, EMID1, FCN1, FMOD,LEFTY1, LEPRE1, MEGF8, MMP1, MMP11, MMP12, MMP2, MMRN2, PLOD2, PLXDC2,PLXND1, S100A11, S100A14, SERPINA3, SERPINF1, SPARC, SVEP1, TGFB1, BMP1,C1QTNF5, FNDC1, COL22A1, GPC4, LOXL2, MUC16, PXDN, SFTPD, and THBS2. Insome embodiments the colon carcinoma ECM protein is not MMP2, SPARC,TGFB1 and/or LOXL2.

The signature of ECM proteins may also be an ECM protein signaturecharacteristic of metastatic primary colon carcinoma. An exemplary ECMprotein signature characteristic of metastatic primary colon carcinomais referred to as GROUP 4 and includes 1 or more of the followingproteins: ADAM10, CLEC11A, FGL2, LOXL1, MFGE8, MMP9, MMRN1, MUC13,PAPLN, PLOD3, PLXNB2, S100A16, ADAM9, ADAMTSL1, C15orf44, CSPG4, EMID1,FCN1, FMOD, LEFTY1, LEPRE1, MEGF8, MMP1, MMP11, MMP12, MMP2, MMRN2,PLOD2, PLXDC2, PLXND1, S100A11, S100A14, SERPINA3, SERPINF1, SPARC,SVEP1, TGFB1, ADAMDEC1, ANXA13, ANXA7, COL8A1, GREM1, INHBE, LEFTY2,LTBP3, LTBP4, MATN2, MFAP4, MUC5B, and THSD4. In some embodiments theECM protein signature characteristic of metastatic primary coloncarcinomas does not include MMP9, MMP2, SPARC and/or TGFB1.

In other embodiments the ECM protein signature characteristic ofmetastatic primary colon carcinoma is referred to as subgroup 4A andinclude 1 or more of the following proteins: ADAM10, CLEC11A, FGL2,LOXL1, MFGE8, MMP9, MMRN1, MUC13, PAPLN, PLOD3, PLXNB2, S100A16, ADAM9,ADAMTSL1, C15orf44, CSPG4, EMID1, FCN1, FMOD, LEFTY1, LEPRE1, MEGF8,MMP1, MMP11, MMP12, MMP2, MMRN2, PLOD2, PLXDC2, PLXND1, S100A11,S100A14, SERPINA3, SERPINF1, SPARC, SVEP1, and TGFB1. In someembodiments the ECM protein signature characteristic of metastaticprimary colon carcinomas does not include: MMP9, MMP2, SPARC and/orTGFB1.

In other embodiments the ECM protein signature characteristic ofmetastatic primary colon carcinoma is referred to as subgroup 4B andincludes 1 or more of the following proteins: ADAM10, CLEC11A, FGL2,LOXL1, MFGE8, MMP9, MMRN1, MUC13, PAPLN, PLOD3, PLXNB2, and S100A16. Insome embodiments the ECM protein characteristic of metastatic primarycolon carcinomas is not MMP9.

In other embodiments the ECM protein signature characteristic ofmetastatic primary colon carcinoma is referred to as subgroup 4C andinclude 1 or more of the following proteins: ADAM9, ADAMTSL1, C15orf44,CSPG4, EMID1, FCN1, FMOD, LEFTY1, LEPRE1, MEGF8, MMP1, MMP11, MMP12,MMP2, MMRN2, PLOD2, PLXDC2, PLXND1, S100A11, S100A14, SERPINA3,SERPINF1, SPARC, SVEP1, and TGFB1. In some embodiments the ECM proteinsignature characteristic of metastatic primary colon carcinoma does notinclude MMP2, SPARC and/or TGFB1.

The signature of ECM proteins may also be an ECM protein signaturecharacteristic of colon carcinoma metastases. The ECM protein signaturecharacteristic of colon carcinoma metastases is a set of proteins thatspecifically identify tumors that have metastasized to a site remotefrom the primary tumor. For instance the tumor may be a liver metastasisof a primary colon tumor. An exemplary ECM protein signaturecharacteristic of colon carcinoma metastases whose expression isassociated with the metastatic process of colon cancer is referred to asGROUP 5 and includes 1 or more of the following proteins: COMP, HPX,IGFALS, SPP1, BMP1, C1QTNF5, FNDC1, ANXA3, C1QC, CCL21, COL10A1,COL15A1, COL27A1, CRLF3, CTSA, CTSH, CXCL12, F12, FCN3, HABP2, IL16,ITIH2, KNG1, LAMA3, LEFTY1, MMP12, MMP7, MST1, NID1, PCOLCE, PRG4,S100A12, S100A4, S100P, SERPINA1, SERPINA3, SERPINB5, SERPINB6,SERPINB9, SERPINC1, SERPIND1, SERPING1, and THBS1.

In other embodiments the ECM protein signature characteristic of coloncarcinoma metastases is referred to as subgroup 5A and includes 1 ormore of the following proteins: COMP, HPX, IGFALS, SPP1, BMP1, C1QTNF5,and FNDC1. In some embodiments the ECM protein signature characteristicof colon carcinoma metastases does not include SPP1.

In other embodiments the ECM protein signature characteristic of coloncarcinoma metastases is referred to as subgroup 5B and includes 1 ormore of the following proteins: COMP, HPX, IGFALS, and SPP1. In someembodiments the ECM protein signature characteristic of colon carcinomametastases does not include SPP1.

In other embodiments the ECM protein signature characteristic of coloncarcinoma metastases is referred to as subgroup 5C and includes 1 ormore of the following proteins: BMP1, C1QTNF5, and FNDC1.

Additionally, the signature of ECM proteins may also be an ECM proteinsignature that specifically marks metastatic colon carcinoma (bothprimary tumors and the secondary tumors or metastases deriving from theprimary tumors) and is referred to herein as of ECM protein signaturecharacteristic of metastatic colon carcinoma (primary & secondary) andas GROUP 6. An exemplary signature of ECM protein signaturecharacteristic of metastatic colon carcinoma (primary & secondary)includes 1 or more of the following proteins: ANXA5, HCFC1, HTRA1,LTBP2, MXRA5, ST14, COL22A1, GPC4, LOXL2, MUC16, PXDN, SFTPD, THBS2,C1QA, C1QB, F2, PLOD1, SERPINB1, TIMP1, AGRN, EFEMP2, HMCN1, SERPINE2,A2M, AEBP1, ANXA1, ANXA11, ANXA4, ANXA6, COL18A1, COL4A1, CSTB, CTSB,CTSD, ECM1, ELANE, F9, FCN1, HRG, IGFBP7, ITIH1, ITIH4, LAMB3, LAMC2,LGALS9, LMAN1, PLG, S100A11, S100A6, S100A8, S100A9, SERPINF1, andSERPINH1. In some embodiments, the ECM protein signature characteristicof metastatic colon carcinoma does not include CTSB and/or S100A8.

In other embodiments the ECM protein signature characteristic ofmetastatic colon carcinoma (primary & secondary) is referred to assubgroup 6A and includes 1 or more of the following proteins: ANXA5,HCFC1, HTRA1, LTBP2, MXRA5, ST14, COL22A1, GPC4, LOXL2, MUC16, PXDN,SFTPD, and THBS2. In some embodiments, the ECM protein signaturecharacteristic of metastatic colon carcinoma does not include LOXL2.

In other embodiments the ECM protein signature characteristic ofmetastatic colon carcinoma (primary & secondary) is referred to assubgroup 6B includes 1 or more of the following proteins: ANXA5, HCFC1,HTRA1, LTBP2, MXRA5, and 5T14

In other embodiments the ECM protein signature characteristic ofmetastatic colon carcinoma (primary & secondary) is referred to assubgroup 6C and includes 1 or more of the following proteins: COL22A1,GPC4, LOXL2, MUC16, PXDN, SFTPD, and THBS2. In some embodiments, the ECMprotein signature characteristic of metastatic colon carcinoma does notinclude LOXL2.

In some embodiments the signature of ECM proteins includes 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,or 112 proteins or at least any of the recited numbers of proteins fromthe above lists.

A signature is a unique group of proteins identifying a particularcharacteristic of a tissue. While one or more of the proteins on each ofthe above lists may have been previously associated with a particulartissue characteristic, the unique combination or signature of each ofthe above lists or sub-lists is novel. Within each list or subgrouppresented above various combinations of more than 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the proteins withineach list or subgroup form a signature.

The full protein sequences for each of the identified ECM proteins areknown in the art. Detailed information on each of these proteinsincluding genomic information, and listing of sources for nucleotide andprotein sequences for each of these proteins is provided in the listsabove in the form of ENTREZ GENE SYMBOL. The full sequences can beidentified by accessing any of these accession numbers in the NationalCenter for Biotechnology Information website: ncbi.nlm.nih.gov. As theseproteins are well known to the skilled artisan, the signatures of ECMproteins and binding reagents described herein include variations andmodifications to the sequences as well. The methods of the inventionresult in the first detailed description of the composition of the tumorextracellular matrix at the protein level and highlight many proteins ofpotential importance for metastatic disease. This information provides aunique opportunity to analyze and characterize the metastatic potentialof tumors. In particular, the findings of the invention can be utilizedto examine changes in metastatic state of tumors with time and/or inresponse to therapeutic interventions. In order to achieve these methodstissue samples may be isolated from a subject at different times with orwithout drug treatments. The different tissue samples can be analyzedfor the presence of the ECM protein or signature proteins. Then thedifferences in ECM protein expression between the two samples can beassessed. The differences can be analyzed qualitatively (e.g. byimmunohistochemistry, IHC) through assessment of the different proteinspresent or quantitatively by measuring levels or approximate levels ofprotein expression. Alternatively the methods of the invention may alsobe performed in vivo without removal of a tissue sample.

The presence of a protein in a tissue sample or the level of a proteinin a tissue sample may be assessed using any known methods in the art.Such methodology is well known. A method commonly used in clinical andpathological assessment is immunohistochemistry, which allowsdetermination of the presence of particular proteins or sets of proteins(such as those in a signature) in a tissue sample. Knowledge ofsignatures allows multiplex qualitative comparisons among tissuesamples, enhancing the diagnostic and prognostic power. The methods ofimmunohistochemistry are well known and widely practiced. When aquantitative assessment of protein levels is made, the levels of proteinmay be compared with either a reference or threshold amount or withamounts found in other samples. For instance, if the presence or levelsof proteins are measured in a primary tumor and its metastasis or acorresponding normal tissue, those can be compared to provide a relativeassessment of the metastatic potential of the tumor. Alternatively thelevels may be compared with an amount that is known (or is shown) to bean amount above or below which a tumor normally expresses the protein.The value that is used in the comparison is referred to as the referenceor threshold level.

The actual numbers in the particular determination of threshold valuesmay vary for different tumors or under different circumstances, such asthe conditions of the assay to determine expression. However, theskilled artisan would be able to identify the correct threshold valuesbased on the circumstances. For example threshold values could easily begenerated using normal non-cancerous tissue under similar circumstances.

The reference sample can be any of a variety of biological samplesagainst which a diagnostic assessment may be made. Examples of referencesamples include biological samples from control populations or controlsamples. Reference samples may be generated through manufacture to besupplied for testing in parallel with the test samples, e.g., referencesample may be supplied in diagnostic kits. Appropriate reference sampleswill be apparent to the skilled artisan.

In other embodiments, the expression level of the protein in the testsample may be determined based on a direct comparison to a referencelevel in absolute values. For instance, at least 10%, at least 20%, atleast 50%, at least 100%, at least 200%, at least 500%, at least 1000%or more higher than the expression level of the protein in the referencesample. In other embodiments, the expression level of the protein in thetest sample is at least 10%, at least 20%, at least 50%, at least 100%,at least 200%, at least 500%, at least 1000% or more lower than theexpression level of the protein in the reference sample.

The presence/absence or levels of proteins or markers may be determinedusing any of a number of techniques available to the person of ordinaryskill in the art for protein analysis, e.g., direct physicalmeasurements (e.g., mass spectrometry), binding assays (e.g.,immunohistochemistry, immunoassays, agglutination assays, andimmunochromatographic assays) etc. The method may also comprisemeasuring a signal that results from a chemical reaction, e.g., a changein optical absorbance, a change in fluorescence, the generation ofchemiluminescence or electrochemiluminescence, a change in reflectivity,refractive index or light scattering, the accumulation or release ofdetectable labels from the surface, the oxidation or reduction or redoxspecies, an electrical current or potential, changes in magnetic fields,etc. Suitable detection techniques may detect binding events bymeasuring the participation of labeled binding reagents through themeasurement of the labels via their photoluminescence (e.g., viameasurement of fluorescence, time-resolved fluorescence, evanescent wavefluorescence, up-converting phosphors, multi-photon fluorescence, etc.),chemiluminescence, electrochemiluminescence, light scattering, opticalabsorbance, radioactivity, magnetic fields, enzymatic activity (e.g., bymeasuring enzyme activity through enzymatic reactions that cause changesin optical absorbance or fluorescence or cause the emission ofchemiluminescence). Alternatively, detection techniques may be used thatdo not require the use of labels, e.g., techniques based on measuringmass (e.g., surface acoustic wave measurements), refractive index (e.g.,surface plasmon resonance measurements), or the inherent luminescence ofan analyte.

Binding assays for measuring protein levels may use solid phase orhomogenous formats. Suitable assay methods include sandwich orcompetitive binding assays. Examples of sandwich immunoassays aredescribed in U.S. Pat. No. 4,168,146 to Grubb et al. and U.S. Pat. No.4,366,241 to Tom et al., both of which are incorporated herein byreference. Examples of competitive immunoassays include those disclosedin U.S. Pat. No. 4,235,601 to Deutsch et al., U.S. Pat. No. 4,442,204 toLiotta, and U.S. Pat. No. 5,208,535 to Buechler et al., all of which areincorporated herein by reference.

Multiple ECM proteins may be measured using a multiplexed assay format,e.g., multiplexing through the use of binding reagent arrays,multiplexing using spectral discrimination of labels, multiplexing byflow cytometric analysis of binding assays carried out on particles(e.g., using the Luminex system). Thus, in some embodiments theinvention encompasses arrays of peptide based detection reagents forassaying 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50or more, or 100 or more binding reagents for binding to the proteinslisted in Groups 1 to 7 otherwise described herein.

Detection of a protein in a test sample involves routine methods. Theskilled artisan can detect the presence or absence of a protein usingwell known methods. Such methods include diverse immunoassays. Ingeneral, immunoassays involve the binding of antibodies or similarprobes to proteins in a sample such a histological section or binding ofproteins in a sample to a solid phase support such as a plastic surface.Detectable antibodies are then added which selectively bind to theprotein of interest. Detection of the antibody indicates the presence ofthe protein. The detectable antibody may be a labeled or an unlabeledantibody. Unlabeled antibody may be detected using a second, labeledantibody that specifically binds to the first antibody or a second,unlabeled antibody which can be detected using labeled protein A, aprotein that complexes with antibodies. Various immunoassay proceduresare described in Immunoassays for the 80's, A. Voller et al., Eds.,University Park, 1981, which is incorporated herein by reference.

Simple immunoassays such as a dot blot and a Western blot involve theuse of a solid phase support which is contacted with a test sample. Anyproteins present in the test sample bind the solid phase support and canbe detected by a specific, detectable antibody preparation. Theintensity of the signal can be measured to obtain a quantitativereadout. Other more complex immunoassays include forward assays for thedetection of a protein in which a first anti-protein antibody bound to asolid phase support is contacted with the test sample. After a suitableincubation period, the solid phase support is washed to remove unboundprotein. A second, distinct anti-protein antibody is then added which isspecific for a portion of the specific protein not recognized by thefirst antibody. The second antibody is preferably detectable. After asecond incubation period to permit the detectable antibody to complexwith the specific protein bound to the solid phase support through thefirst antibody, the solid phase support is washed a second time toremove the unbound detectable antibody. Alternatively, in a forwardsandwich assay a third detectable antibody, which binds the secondantibody is added to the system. Other types of immunometric assaysinclude simultaneous and reverse assays. A simultaneous assay involves asingle incubation step wherein the first antibody bound to the solidphase support, the second, detectable antibody and the test sample areadded at the same time. After the incubation is completed, the solidphase support is washed to remove unbound proteins. The presence ofdetectable antibody associated with the solid support is then determinedas it would be in a conventional assays. A reverse assay involves thestepwise addition of a solution of detectable antibody to the testsample followed by an incubation period and the addition of antibodybound to a solid phase support after an additional incubation period.The solid phase support is washed in conventional fashion to removeunbound protein/antibody complexes and unreacted detectable antibody.

A number of methods are well known for the detection of antibodies. Forinstance, antibodies can be detectably labeled by linking the antibodiesto an enzyme and subsequently using the antibodies in an enzymeimmunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA), such asa capture ELISA. The enzyme, when subsequently exposed to its substrate,reacts with the substrate and generates a chemical moiety which can bedetected, for example, by spectrophotometric, fluorometric or visualmeans. Enzymes which can be used to detectably label antibodies include,but are not limited to malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

A detectable label is a moiety, the presence of which can be ascertaineddirectly or indirectly. Generally, detection of the label involves anemission of energy by the label. The label can be detected directly byits ability to emit and/or absorb photons or other atomic particles of aparticular wavelength (e.g., radioactivity, luminescence, optical orelectron density, etc.). A label can be detected indirectly by itsability to bind, recruit and, in some cases, cleave another moiety whichitself may emit or absorb light of a particular wavelength (e.g.,epitope tag such as the FLAG epitope, enzyme tag such as horseradishperoxidase, etc.). An example of indirect detection is the use of afirst enzyme label which cleaves a substrate into visible products. Thelabel may be of a chemical, peptide or nucleic acid molecule naturealthough it is not so limited. Labels include any known labels that canbe used with imaging techniques, such as PET isotopes, scintigraphy,NMR, etc. Other detectable labels include radioactive isotopes such asP³² or H³, luminescent markers such as fluorochromes, optical orelectron density markers, etc., or epitope tags such as the FLAG epitopeor the HA epitope, biotin, avidin, and enzyme tags such as horseradishperoxidase, β-galactosidase, nanoparticles, etc. The label may be boundto a reagent during or following its synthesis. There are many differentlabels and methods of labeling known to those of ordinary skill in theart. Examples of the types of labels that can be used in the presentinvention include enzymes, radioisotopes, fluorescent compounds,colloidal metals, nanoparticles, chemiluminescent compounds, andbioluminescent compounds. Those of ordinary skill in the art will knowof other suitable labels for the reagents described herein, or will beable to ascertain such, using routine experimentation. Furthermore, thecoupling or conjugation of these labels to the reagents of the inventioncan be performed using standard techniques common to those of ordinaryskill in the art.

Another labeling technique which may result in greater sensitivityconsists of coupling the molecules described herein to low molecularweight haptens. These haptens can then be specifically altered by meansof a second reaction. For example, it is common to use haptens such asbiotin, which reacts with avidin, or dinitrophenol, pyridoxal, orfluorescein, which can react with specific anti-hapten antibodies.

Conjugation of the reagents including antibodies or fragments thereof toa detectable label facilitates, among other things, the use of suchagents in diagnostic assays. Another category of detectable labelsincludes diagnostic and imaging labels (generally referred to as in vivodetectable labels) such as for example magnetic resonance imaging (MRI):Gd(DOTA); for nuclear medicine: ²⁰¹Tl, gamma-emitting radionuclide^(99m)Tc; for positron-emission tomography (PET): positron-emittingisotopes, (18)F-fluorodeoxyglucose ((18)FDG), (18)F-fluoride, copper-64,gadodiamide, and radioisotopes of Pb(II) such as 203Pb; 111In.

As used herein, “conjugated” means two entities stably bound to oneanother by any physiochemical means. It is important that the nature ofthe attachment is such that it does not impair substantially theeffectiveness of either entity. Keeping these parameters in mind, anycovalent or non-covalent linkage known to those of ordinary skill in theart may be employed. In some embodiments, covalent linkage is preferred.Noncovalent conjugation includes hydrophobic interactions, ionicinteractions, high affinity interactions such as biotin-avidin andbiotin-streptavidin complexation and other affinity interactions. Suchmeans and methods of attachment are well known to those of ordinaryskill in the art.

Thus, the invention contemplates ECM protein binding reagents which mayoptionally be conjugated to a detectable label for use in the methods ofthe invention. In other aspects, the invention contemplates ECM proteinbinding reagents conjugated to an active agent. The active agent may bea detectable label as described above. Such compounds are useful invitro or in vivo for detecting and characterizing tumor cells. Theactive agent may also be a drug or therapeutic such as an anti-cancerdrug. Such compounds may be used as therapeutic conjugates to treattumors.

The therapeutic conjugates include a binding reagent such as an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g. an enzymatically active toxin of bacterial, fungal, plant oranimal origin, or fragments thereof, or a small molecule toxin), or aradioactive isotope (i.e., a radioconjugate). Other antitumor agentsthat can be conjugated to the binding reagents of the invention includeBCNU, streptozoicin, vincristine and 5-fluorouracil, the family ofagents known collectively LL-E33288 complex described in U.S. Pat. Nos.5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296).Enzymatically active toxins and fragments thereof which can be used inthe conjugates include diphtheria A chain, nonbinding active fragmentsof diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI,PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin,sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin,phenomycin, enomycin and the tricothecenes.

For selective destruction of the cell, the antibody may be conjugated ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples include²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ²¹²Pb andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for example^(99m)Tc or ¹²³I, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such as ¹²³I,¹³¹I, ¹¹¹In, ¹⁹F, ¹³C, ¹⁵N, 17O, Gadolinium, Manganese or Iron. Theradio- or other labels may be incorporated in the conjugate in knownways. For example, the reagent may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as ^(99m) Tc or ¹²³I, ¹⁸⁶Re, ¹⁸⁸Re and ¹¹¹In can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

Additionally, cytokines and antibody cytokine conjugates can be usedtherapeutically. Antibodies to the ECM proteins may be used to delivercytokines which can have a number of functions including enhancing animmune response to the cancerous tissue. Cytokines include but are notlimited to IL-2, IL-6, IL-8, IL-10, IL-12, IL-18, TNF, IFN-γ, IFN-β,chemokines, and IFN-α.

In one aspect, the invention provides methods for the treatment ofcancer. The terms “tumor”, “cancer”, “cancerous tissue” and “carcinoma”are used interchangeably herein, and each, refers to an uncontrolledgrowth of cells which interferes with the normal functioning of thebodily organs and systems. Cancers, including those cancers whichmigrate from their original location and seed vital organs, caneventually lead to the death of the subject through the functionaldeterioration of the affected organs. Cancers can be classified into avariety of categories including, carcinomas, sarcomas and hematopoieticcancers. Carcinomas are malignant cancers that arise from epithelialcells and include adenocarcinoma and squamous cell carcinoma.

As used herein, a subject is a human, non-human primate, cow, horse,pig, sheep, goat, dog, cat, or rodent. In all embodiments human subjectsare preferred. In aspects of the invention pertaining to predictivetherapy in cancers, the subject is a human either suspected of havingthe cancer, or having been diagnosed with cancer. Methods foridentifying subjects suspected of having cancer may include physicalexamination, subject's family medical history, subject's medicalhistory, biopsy, or a number of imaging technologies such asultrasonography, computed tomography, magnetic resonance imaging,magnetic resonance spectroscopy, or positron emission tomography.Diagnostic methods for cancer and the clinical delineation of cancerdiagnoses are well known to those of skill in the medical arts.

In some aspects the specific ECM proteins identified herein may beutilized as a therapeutic target. These proteins can be targeted byspecific reagents designed to interfere with their pro-metastaticfunctions and or expression. For example many of these proteins havespecific receptors and therapeutic agents can be used to block theinteractions of ECM proteins with their receptors or with other ECMproteins in order to treat cancer. Additionally, some of the ECMproteins are enzymes. Therapeutics may be used to interfere with theenzymatic activities of these proteins. Additionally, the expression ofthese proteins can be inhibited using inhibitory RNA, particularly whenthe RNA can be targeted to the tumor tissue. A therapeutic agent usefulfor blocking a protein-receptor or a protein-protein interaction is anytype of reagent that binds to one or both of the proteins (receptor orligand) and blocks the proteins from interacting. The reagent may be aprotein, small molecule, nucleic acid or any other type of moleculewhich binds to and blocks the interaction, such as a receptorantagonist. For example the reagent may be (using antibodies, antibodyfragments, peptides or peptidomimetics. Integrins as therapeuticreagents are described in, for example, Goodman and Picard, TIPS, 968,p. 1 (2012). Additionally anti-integrins, such as anti-integrinantibodies may be used as therapeutic reagents.

A therapeutic agent useful for blocking enzyme function is any reagentthat interrupts the interaction or activity of the enzyme with it'ssubstrate. For example the reagent may directly interfere with theinteraction. For instance a structural antagonist of the substrate maycompete for binding to the enzyme and block the interaction between theenzyme and substrate. Additionally the regent may indirectly interferewith the interaction by causing a conformational change or stabilitychange in the enzyme which results in a loss of the enzymes ability tobind to the substrate or act on the substrate.

Methods for inhibiting the expression of genes encoding ECM proteinsdescribed herein are known in the art. For example, gene knockdownstrategies may be used that make use of RNA interference (RNAi) and/ormicroRNA (miRNA) pathways including small interfering RNA (siRNA), shorthairpin RNA (shRNA), double-stranded RNA (dsRNA), miRNAs, and othersmall interfering nucleic acid-based molecules known in the art. In oneembodiment, vector-based RNAi modalities (e.g., shRNA or shRNA-mirexpression constructs) are used to reduce expression of a gene encodingany of the ECM proteins described herein.

Examples of specific inhibitors of the ECM proteins described hereininclude, but are not limited to the inhibitors shown for each protein inthe following Table.

PLAT (tPA) PAI-1, Ab-1 available commercially from Millipore or sodiumphenylbutyrate MMP1 GM6001 MMP Inhibitor available commercially fromMilliporeenzyme inhibitors exist candoxatril MMP2 anti-MMP-2 proformantibody available from commercially from Millipore; Candoxatril CTSB a2macroglobulin MMP19 Candoxatril EGLN1 series of imidazo[1,2-a]pyridinederivatives cited in US2009176726A) F10 NSC128437OIUPAC:4-(4-cyclohexylamino-9,10-dioxo- anthracen-1-yl)aminobenze-ne-sulfonicacid NSC125908PIUPAC: 3-(9,10-dioxo-2-sulfo-anthracen-1-yl)diazenyl-2-hydroxy-b-enzoicacid NSC9600QIUPAC:4-[N′-(2-hydroxynaphthalen-1-yl)-N′-sulfo-hydrazino]benzene-sulfonicacid NSC13778JIUPAC:3-(3-stibonophenyl)prop-2-enoicacid 1NSC119110-U, NSC119111-V,NSC119913-X, NSC119915-Z, NSC170008-Y, and NSC306711-P) or (e.g.,benthiavalicarb, dimethomorph, flumorph, iprovalicarb, mandipropamid,polyoxins, polyoxorim, prothiocarb, validamycin A, and valiphenal) F13bEnzymatic inhibitors disclosed in US4218476A Antibodies disclosed inU.S. Pat. No. 5,620,688 Vwf Sulfobacins A and B, Anti-von WillebrandFactor Therapeutic Aptamer ARC1779 LOX inhibitor may be, for example, anantibody against LOX LOXL1 or LOXL, a small molecule inhibitor, siRNA,shRNA or LoXL2 an antisense polynucleotide against LOX or LOXL. Theinhibitors may be noncompetitive inhibitors. (e.g. BAPN, pAPN)[AU2008282739AA] [Antibodies] (e.g., anti- LOXL2 monoclonal antibodyGS-6634) MMP9 (e.g., Anti-MMP-9 Antibody nzyme inhibitors exist MMP11candoxatril MMP12 candoxatril PLOD2 Anti-PLOD2 ADAMDEC1 Tamoxifenavailable commercially from Nolvadex MMP12 MMP-12 Inhibitor, MMP408;Anti-MMP-12 (C-terminus) Antibody and Anti-TIMP-1 Antibody AB770available from commercially from Millipore; candoxatril F2 sulodexide,ximelagatran, warfarin, phenprocoumon, enoxaparin, ardeparin,fondaparinux, latamoxef, bacitracin, ticlopidine and erdosteine CTSDSerine Dependent Enzymes: These include such enzymes as Elastase (humanleukocyte), Cathepsin G, Thrombin, Plasmin, C-1 Esterase,C-3-Convertase, Urokinase, Plasminogen activator, Acrosin, B-Lactamase,D-Alanine- D-Alanine Carboxypeptidase, Chymotrypsin, Trypsin andkallikreins. Thiol Dependent Enzymes: Cathepsin B. Carboxylic AcidDependent Enzymes: These include such specific enzymes as Renin, Pepsinand Cathepsin D. Metallo Dependent Enzymes: These include AngiotensineConverting Enzyme, Enkephalinase, Pseudomonas Elastase and LeucineAminopeptidase. (e.g. the compounds: CBZ-Val-Val-Phe-CF2-CO-Ala-IaaCBZ-Val-Val-Phe-CF2H, CBZ-Val-Val-Phe-CF3,CBZ-Val-Val-PheICF2-PheIAla-WH(CH2)2CH(cH3)2CBZ-Val-Val-PhetCF2-Phe]Ala-NHcH2cH(cH3)2 Iaa being isoamyl amide F9TAP, FIX, IXai PLG aminocaproic acid

When used in combination with the therapies of the invention the dosagesof known therapies may be reduced in some instances, to avoid sideeffects.

Cancer therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in suchliterature as the Physician's Desk Reference (56^(th) ed., 2002). Insome embodiments, the therapeutic compounds of the invention areformulated into a pharmaceutical composition that further comprises oneor more additional anticancer agents.

The active agents of the invention are administered to the subject in aneffective amount for treating the subject. An “effective amount”, forinstance, is an amount necessary or sufficient to realize a desiredbiologic effect. For instance an effective amount is that amountsufficient to prevent or inhibit cancer cell growth or proliferation oralternatively an amount sufficient to induce apoptosis of a cancer cellor induce tumor regression. In some preferred embodiments the effectiveamount is that amount useful for reducing the development of metastaticcancers.

The effective amount of a compound of the invention in the treatment ofa subject may vary depending upon the specific compound used, the modeof delivery of the compound, and whether it is used alone or incombination. The effective amount for any particular application canalso vary depending on such factors as the type and/or degree of cancerin a subject, the particular compound being administered for treatment,the size of the subject, or the severity of the disorder. One ofordinary skill in the art can empirically determine the effective amountof a particular molecule of the invention without necessitating undueexperimentation. Combined with the teachings provided herein, bychoosing among the various active compounds and weighing factors such aspotency, relative bioavailability, patient body weight, severity ofadverse side-effects and preferred mode of administration, an effectiveprophylactic or therapeutic treatment regimen can be planned which doesnot cause substantial toxicity in and of itself and yet is entirelyeffective to treat the particular subject.

Toxicity and efficacy of the protocols of the present invention can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Prophylactic and/or therapeutic agents that exhibit largetherapeutic indices are preferred. While prophylactic and/or therapeuticagents that exhibit toxic side effects may be used, care should be takento design a delivery system that targets such agents to the site ofaffected tissue in order to minimize potential damage to uninfectedcells and, thereby, reduce side effects.

The data obtained from the cell culture assays, animal studies and humanstudies can be used in formulating a range of dosage of the prophylacticand/or therapeutic agents for use in humans. The dosage of such agentslies preferably within a range of circulating concentrations thatinclude the ED50 with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any agent used in the method of theinvention, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose may be formulated in animal models toachieve a circulating plasma concentration range that includes the IC50(i.e., the concentration of the test compound that achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

As used herein, the term treat, treated, or treating when used withrespect to a disorder refers to a prophylactic treatment which increasesthe resistance of a subject to development of the disease or, in otherwords, decreases the likelihood that the subject will develop thedisease as well as a treatment after the subject has developed thedisease in order to fight the disease, prevent the disease from becomingworse, or slow the progression of the disease compared to in the absenceof the therapy.

The diagnostic and therapeutic compounds described herein can beadministered in combination with other therapeutic agents and suchadministration may be simultaneous or sequential. When the othertherapeutic agents are administered simultaneously they can beadministered in the same or separate formulations, but are administeredat the same time. The administration of the other therapeutic agent,including chemotherapeutics can also be temporally separated, meaningthat the therapeutic agents are administered at a different time, eitherbefore or after, the administration of the therapeutics describedherein. The separation in time between the administration of thesecompounds may be a matter of minutes or it may be longer.

Thus, in some instances, the invention also involves administeringanother cancer treatment (e.g., radiation therapy, chemotherapy orsurgery) to a subject. Examples of conventional cancer therapies includetreatment of the cancer with agents such as All-trans retinoic acid,Actinomycin D, Adriamycin, anastrozole, Azacitidine, Azathioprine,Alkeran, Ara-C, Arsenic Trioxide (Trisenox), BiCNU Bleomycin, Busulfan,CCNU, Carboplatin, Capecitabine, Cisplatin, Chlorambucil,Cyclophosphamide, Cytarabine, Cytoxan, DTIC, Daunorubicin, Docetaxel,Doxifluridine, Doxorubicin, 5-flurouracil, Epirubicin, Epothilone,Etoposide, exemestane, Erlotinib, Fludarabine, Fluorouracil,Gemcitabine, Hydroxyurea, Herceptin, Hydrea, Ifosfamide, Irinotecan,Idarubicin, Imatinib, letrozole, Lapatinib, Leustatin, 6-MP,Mithramycin, Mitomycin, Mitoxantrone, Mechlorethamine, megestrol,Mercaptopurine, Methotrexate, Mitoxantrone, Navelbine, Nitrogen Mustard,Oxaliplatin, Paclitaxel, pamidronate disodium, Pemetrexed, Rituxan,6-TG, Taxol, Topotecan, tamoxifen, taxotere, Teniposide, Tioguanine,toremifene, trimetrexate, trastuzumab, Valrubicin, Vinblastine,Vincristine, Vindesine, Vinorelbine, Velban, VP-16, and Xeloda.

Multiple doses of the molecules of the invention are also contemplated.In some instances, when the molecules of the invention are administeredwith another therapeutic, for instance, a chemotherapeutic agent asub-therapeutic dosage of either or both of the molecules may be used. A“sub-therapeutic dose” as used herein refers to a dosage which is lessthan that dosage which would produce a therapeutic result in the subjectif administered in the absence of the other agent.

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more agents, dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. Moreover, for animal (e.g., human) administration, itwill be understood that preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biological Standards. The compounds are generally suitable foradministration to humans. This term requires that a compound orcomposition be nontoxic and sufficiently pure so that no furthermanipulation of the compound or composition is needed prior toadministration to humans.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences(1990), incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the therapeutic or pharmaceutical compositions is contemplated. Thecompounds may be sterile or non-sterile.

The compounds described herein may comprise different types of carriersdepending on whether it is to be administered in solid, liquid oraerosol form, and whether it need to be sterile for such routes ofadministration as injection. The present invention can be administeredintravenously, intradermally, intraarterially, intralesionally,intratumorally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, intraperitoneally, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularally, orally, topically, locally, inhalation (e.g., aerosolinhalation), injection, infusion, continuous infusion, localizedperfusion bathing target cells directly, via a catheter, via a lavage,in creams, in lipid compositions (e.g., liposomes), or by other methodor any combination of the forgoing as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences(1990), incorporated herein by reference). In a particular embodiment,intraperitoneal injection is contemplated.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more components. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

The agent may be formulated into a composition in a free base, neutralor salt form. Pharmaceutically acceptable salts, include the acidaddition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups also can be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

The compounds of the invention may be administered directly to a tissue.Direct tissue administration may be achieved by direct injection. Thecompounds may be administered once, or alternatively they may beadministered in a plurality of administrations. If administered multipletimes, the compounds may be administered via different routes. Forexample, the first (or the first few) administrations may be madedirectly into the affected tissue while later administrations may besystemic.

The formulations of the invention are administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, adjuvants, and optionally other therapeuticingredients. In general, a pharmaceutical composition comprises thecompound of the invention and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers for nucleic acids, small molecules,peptides, monoclonal antibodies, and antibody fragments are well-knownto those of ordinary skill in the art. As used herein, apharmaceutically acceptable carrier means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients.

Pharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers and other materials which arewell-known in the art. Exemplary pharmaceutically acceptable carriersfor peptides in particular are described in U.S. Pat. No. 5,211,657.Such preparations may routinely contain salt, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents. When used in medicine, the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically acceptable salts thereof and are notexcluded from the scope of the invention. Such pharmacologically andpharmaceutically acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

The compounds of the invention may be formulated into preparations insolid, semi-solid, liquid or gaseous forms such as tablets, capsules,powders, granules, ointments, solutions, depositories, inhalants andinjections, and usual ways for oral, parenteral or surgicaladministration. The invention also embraces pharmaceutical compositionswhich are formulated for local administration, such as by implants,including those designed for slow or controlled release.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active agent. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids, such as a syrup,an elixir or an emulsion.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a subject to be treated. Pharmaceutical preparations fororal use can be obtained as solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Optionally the oralformulations may also be formulated in saline or buffers forneutralizing internal acid conditions or may be administered without anycarriers.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch. Techniques forpreparing aerosol delivery systems are well known to those of skill inthe art. Generally, such systems should utilize components which willnot significantly impair the biological properties of the active agent(see, for example, Sciarra and Cutie, “Aerosols,” in Remington'sPharmaceutical Sciences, 18th edition, 1990, pp 1694-1712; incorporatedby reference). Those of skill in the art can readily determine thevarious parameters and conditions for producing aerosols without resortto undue experimentation.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Lower doses will result from other forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of compounds.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the agents of the invention to the subject. Biodegradablematrices are preferred. Such polymers may be natural or syntheticpolymers. Synthetic polymers are preferred. The polymer is selectedbased on the period of time over which release is desired, generally inthe order of a few hours to a year or longer. Typically, release over aperiod ranging from between a few hours and three to twelve months ismost desirable. The polymer optionally is in the form of a hydrogel thatcan absorb up to about 90% of its weight in water and further,optionally is cross-linked with multivalent ions or other polymers.

The invention also encompasses binding reagents capable of interactingwith ECM proteins, optionally in the form of conjugates, as describedabove. In some embodiments the binding reagent is an ECM protein bindingreagent. The ECM protein binding reagents of the invention bind to anECM protein, preferably in a selective manner. As used herein, the terms“selective binding” and “specific binding” are used interchangeably withrespect to reagents to refer to the ability of the reagent to bind withgreater affinity to ECM proteins and fragments thereof than to non-ECMproteins. That is, reagents that bind selectively to ECM proteins willnot bind to non-ECM proteins to the same extent and with the sameaffinity as they bind to ECM proteins and fragments thereof. In someembodiments, the reagents of the invention bind solely to ECM proteinsand fragments thereof. As used herein, a binding reagent that bindsselectively or specifically to ECM proteins will bind with lesseraffinity (if at all) to non-ECM proteins or other different ECMproteins. Lesser affinity may include at least 10% less, 20% less, 30%less, 40% less, 50% less, 60% less, 70% less, 80% less, 90% less, or 95%less.

The binding reagents useful according to the invention are isolated andinclude but are not limited to small molecules, isolated peptides,isolated antibodies and isolated antibody fragments. “Isolated peptides”as used herein refer to peptides that are substantially physicallyseparated from other cellular material (e.g., separated from cells whichproduce the antibodies) or from other material that hinders their useeither in the diagnostic or therapeutic methods of the invention.

The term “antibody” herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, antibody fragments, so long as they exhibitthe desired biological activity, and antibody like molecules such asscFv. A native antibody usually refers to heterotetrameric glycoproteinscomposed of two identical light (L) chains and two identical heavy (H)chains. Each heavy and light chain has regularly spaced intrachaindisulfide bridges. Each heavy chain has at one end a variable domain(VH) followed by a number of constant domains. Each light chain has avariable domain at one end (VL) and a constant domain at its other end;the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light-chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light- andheavy-chain variable domains.

ECM protein-antibodies are available commercially, from numerous sourcesincluding companies such as Abcam, AbD Serotec, Abnova, ThermoScientific, Pierce Antibodies, Advanced Targeting Systems, Novus Bio, BDPharmingen and many others. The commercial antibodies may be used as isor modified or humanized by methods well known to the skilled artisan.

The invention also includes kits made up of the various reagentsdescribed herein assembled to accomplish the methods of the invention. Akit for instance may include one or more reagents for detecting one ormore ECM proteins or signatures of ECM proteins. The kit may furthercomprise assay diluents, standards, controls and/or detectable labels.The assay diluents, standards and/or controls may be optimized for aparticular sample matrix. Reagents include, for instance, antibodies,nucleic acids, labeled secondary agents, or in the alternative, if theprimary reagent is labeled, enzymatic or agent binding reagents whichare capable of reacting with the labeled reagent. One skilled in the artwill readily recognize that reagents of the present invention can bereadily incorporated into one of the established kit formats which arewell known in the art.

As used herein, “promoted” includes all methods of doing businessincluding methods of education, hospital and other clinical instruction,pharmaceutical industry activity including pharmaceutical sales, and anyadvertising or other promotional activity including written, oral andelectronic communication of any form, associated with compositions ofthe invention in connection with treatment or characterization of acancer.

“Instructions” can define a component of promotion, and typicallyinvolve written instructions on or associated with packaging ofcompositions of the invention. Instructions also can include any oral orelectronic instructions provided in any manner.

Thus the agents described herein may, in some embodiments, be assembledinto pharmaceutical or diagnostic or research kits to facilitate theiruse in therapeutic, diagnostic or research applications. A kit mayinclude one or more containers housing the components of the inventionand instructions for use. Specifically, such kits may include one ormore agents described herein, along with instructions describing theintended therapeutic application and the proper administration of theseagents. In certain embodiments agents in a kit may be in apharmaceutical formulation and dosage suitable for a particularapplication and for a method of administration of the agents.

The kit may be designed to facilitate use of the methods describedherein by physicians and can take many forms. Each of the compositionsof the kit, where applicable, may be provided in liquid form (e.g., insolution), or in solid form, (e.g., a dry powder). In certain cases,some of the compositions may be constitutable or otherwise processable(e.g., to an active form), for example, by the addition of a suitablesolvent or other species (for example, water or a cell culture medium),which may or may not be provided with the kit. As used herein,“instructions” can define a component of instruction and/or promotion,and typically involve written instructions on or associated withpackaging of the invention. Instructions also can include any oral orelectronic instructions provided in any manner such that a user willclearly recognize that the instructions are to be associated with thekit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet,and/or web-based communications, etc. The written instructions may be ina form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, whichinstructions can also reflect approval by the agency of manufacture, useor sale for human administration.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample and applying to a subject. The kit mayinclude a container housing agents described herein. The agents may beprepared sterilely, packaged in syringe and shipped refrigerated.Alternatively it may be housed in a vial or other container for storage.A second container may have other agents prepared sterilely.Alternatively the kit may include the active agents premixed and shippedin a syringe, vial, tube, or other container.

The following examples are provided to illustrate specific instances ofthe practice of the present invention and are not intended to limit thescope of the invention. As will be apparent to one of ordinary skill inthe art, the present invention will find application in a variety ofcompositions and methods.

EXAMPLES

Methods

Tissue Preparation and ECM Protein Enrichment—

Sequential extractions of frozen samples of tissues or tumors wereperformed using the CNMCS (Cytosol/Nucleus/Membrane/Cytoskeleton)Compartmental Protein Extraction kit (Cytomol, Union City, Calif.)according to manufacturer's instructions. In brief, frozen tissues(150-200 mg) or tumors (200-j400 mg) were homogenized and extractedsequentially to remove (1) cytosolic proteins (2), nuclear proteins (3),membrane proteins (4), and cytoskeletal proteins leaving a finalinsoluble fraction enriched for ECM proteins. Fractions were separatedon SDS-polyacrylamide gradient gels, transferred to nitrocellulosemembranes and probed with antibodies to proteins characteristic ofdifferent subcellular compartments.

Mass Spectrometry—

ECM-enriched fractions were solubilized in urea, disulfide bonds reducedand alkylated, and proteins digested with PNGaseF, Lys-C, and trypsin.Solutions that began cloudy upon initial reconstitution were clear afterovernight digestion. The resulting peptides were separated by off-gelelectrophoresis (OGE) according to isoelectric point and byreversed-phase high-performance liquid chromatography followed by tandemmass spectrometry (MS/MS) on an LTQ Orbitrap mass spectrometer. Massspectra were interpreted with SpectrumMill and annotated using thematrisome bioinformatics lists developed in this work. MS/MS spectrawere searched against a UniProt database containing either mouse only orboth mouse (53,448 entries) and human (78,369 entries) sequences; allsequences (including isoforms and excluding fragments) were downloadedfrom the UniProt web site. To each database a set of common laboratorycontaminant proteins (73 entries) was appended. Peptides identified witha false discovery rate <2.5% were assembled into identified proteins,and an in silico protein list was then used to categorize all of theidentified proteins as being ECM derived or not. MS/MS spectra searchesallowed for carbamidomethylation of cysteines and possible carbamylationof N termini as fixed/mix modifications. Allowed variable modificationswere oxidized methionine, deamidation of asparagine, pyro-glutamic acidmodification at N-terminal glutamine, and hydroxylation of proline witha precursor shift range of −18 to 97 Da.

Bioinformatic Definition of Extracellular Matrix Proteins—

The human and mouse proteomes were each screened for proteins containingdomains characteristic of ECM proteins, ECM-affiliated proteins, ECMmodifiers and secreted factors. Those lists were subsequently screenedto eliminate proteins that shared one or more of the defining domainsbut were not ECM or ECM-associated proteins based on other criteria. Awebpage providing collection of resources (data files, sequence files)and further annotations on the bioinformatic pipeline developedaccording to these methods has been set upweb.mit.edu.hyneslab/matrisome/. This site contains more detailedinformation on the methodology and is incorporated by reference.

Immunohistochemistry—

Tumor samples were formalin-fixed and paraffin-embedded. Sections weredewaxed and rehydrated following standard procedures. Antigen retrievalwas performed by standard known methods. Sections were then blocked withPBS. Incubation with antibody was performed and secondary antibodyincubation. Sections were counterstained to visualize nuclei.

Results:

ECM Protein Enrichment From Tissues—

Analysis of the protein composition of the extracellular matrix presentschallenges due to the diversity, large size, insolubility andcrosslinking of these proteins. By contrast, most other cellularcomponents are soluble even at relatively low concentrations of salt ordetergents. Therefore, we took advantage of the insolubility of ECMproteins to enrich for them while depleting other cellular components.We used a subcellular fractionation protocol to extract sequentiallycomponents from the cytosol, the nucleus, the membrane and thecytoskeleton and enrich for ECM proteins. ECM proteins such asfibronectin were not extracted during these intermediate steps and werefound to be enriched in the final insoluble fraction.

Development of a Proteomics-based Strategy to Characterize theComposition of ECM In Vivo—

To analyze the composition of the ECM-enriched fractions obtained afterdepletion of other cellular components, we digested the proteins topeptides and employed a proteomics pipeline using liquid chromatographycombined with tandem mass spectrometry (LC-MS/MS) to identify peptidesand proteins. Analysis by LC-MS/MS of ECM proteins enriched and digestedto peptides confirmed a significant enrichment for matrix proteins, withmore than 75% of the total precursor ion intensity (the sum of MS1precursor ion peak areas for all identified peptides) corresponding toproteins defined as ECM. To help measure the success of our enrichmentstrategy and focus downstream biological follow-up we sought tocategorize the identified proteins as being ECM-derived or not. Thecategorization of each protein identified by mass spectrometry wasinitially performed using the Gene Ontology (GO) “Cellular Compartment”annotations. However, this annotation showed several clear limitations.In order to interpret the mass spectrometric data we needed a betterdefinition of which proteins should be considered as part of the ECM.

In Silico Definition of the ECM Proteins—

A Bioinformatic approach was developed to predict within any genome theensemble of genes encoding what we define as the “matrisome,” namely allthose components constituting the extracellular matrix (the “corematrisome”) and those components associated with it(“matrisome-associated” proteins). One hallmark of ECM proteins is theirdomain-based structure. Exploiting this characteristic, we established alist of 55 diagnostic InterPro domains commonly found in ECM proteins(type I, II and III fibronectin domains, type I thrombospondin repeats,laminin G domain, etc). This domain list was used to screen the UniProtprotein database. We know that some of the domains used to selectpositively for ECM proteins are also found in transmembrane receptorsand proteins involved in cell adhesion (growth factor receptors,integrins, etc) that do not belong to the ECM. These families ofproteins also display a subset of specific domains (e.g. tyrosine kinaseand phosphatase domains) and transmembrane domains incompatible withdefinition as “extracellular matrix” proteins. Therefore, a second stepcomprising a negative selection using 20 domains and a transmembranedomain prediction was used. This analysis was performed in parallel forboth the mouse and human genomes and the respective murine and humanmatrisome lists were compared based on orthology. Manual curation of thematrisome lists also allowed us to add a very few known ECM proteinsthat do not contain any known domains; for example, dermatopontin anddentin sialophosphoprotein. Finally, knowledge-based annotation of thesegene lists allowed us to define subcategories within the core matrisome;namely, ECM glycoproteins, collagens, and proteoglycans. The definedsets of core matrisome proteins are described herein and included in thesignatures defined above.

Breast Cancer Analysis

Poorly metastatic (MDA-MB-231) or highly metastatic (LM2) human mammarycarcinoma cells were orthotopically injected in immune-deficient miceNOD/SCID/IL2Rγ^(−/−). The tumors formed were resected 6 weekspost-injection and the extracellular matrix of these tumors wasextracted. The composition of the ECMs was determined for 2 independenttumors for each tumor type, by mass spectrometry. The comparison of theECM composition of poorly and highly metastatic mammary tumor xenograftsled to the definition of ECM signatures of each tumor type—GROUPS 1-3.

Colon Cancer Analysis

A panel of three samples: normal colon mucosa, colon tumor and livermetastasis was obtained for each of three human patients. Tocharacterize the composition of the normal liver extracellular matrix(ECM), two pools of samples were generated from 4 and 5 healthy patientsrespectively. The ECM of these tissues and tumors was extracted and thecomposition of the ECMs was determined by mass spectrometry.

The comparison of the ECM composition of normal vs. tumor samples led tothe identification of:

1) a set of proteins only detected in colon tumors (and in at least ⅔patients) and not in any other tissues (normal colon, normal liver orliver met. samples)—GROUP 4

2) a set of proteins only detected in liver metastases (and in at least⅔ patients) and not in any other tissues (normal colon, normal liver orcolon tumor samples)—GROUP 5

3) an additional set of proteins was detected in at least ⅔ colon tumorsand at least ⅔ liver metastases but not in the normal tissues.—GROUP 6

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

What is claimed is:
 1. A method of delivering an active agent to a tumor, comprising administering to a subject having a tumor that is a mammary carcinoma, a binding reagent which interacts specifically with an extracellular matrix (ECM) protein, wherein the binding reagent is conjugated to an active agent in an effective amount to deliver the active agent to the tumor, wherein the ECM protein is characteristic of mammary carcinoma and is selected from a signature of ECM proteins, referred to as Group A, comprising COL17A1, COL19A1, Col28a1, COL6A6, EMID2, FLG2, Gm7455, Hmcn1, HRNR, Itih3, NGLY1, PRELP, Vwa1, AGRN, C1qc, COL22A1, COL23A1, COL24A1, EFEMP2, F10, F13b, HCFC2, HTRA1, Itih4, LEPREL2, LTBP3, MFGE8, P4HTM, Pap1n, Plxnb2, Serpina1b, Serpinf1, SNED1, SRPX, and TINAGL1.
 2. The method of claim 1, wherein the binding reagent is an antibody or an antibody fragment.
 3. The method of claim 1, wherein the active agent is a detectable label or a chemotherapeutic agent.
 4. The method of claim 1, wherein the method involves multiplex comparisons among tissue samples.
 5. The method of claim 1, wherein the detectable label is a diagnostic or imaging in vivo detectable label.
 6. The method of claim 1, wherein the imaging in vivo detectable label is selected from Gd(DOTA), 201Tl, gamma-emitting radionuclide 99mTc; positron-emitting isotopes, (18)F-fluorodeoxyglucose ((18)FDG), (18)F-fluoride, copper-64, gadodiamide, and radioisotopes of Pb(II) such as 203Pb; 111In. 